The hypocretin (Hcrt)-expressing neurons of the hypothalamus and the noradrenergic (NA) neurons of the locus ceruleus (LC) in the brainstem form two major centers of sleep regulation. Activity in these centers is correlated with wakefulness and stimulation of either cluster increases the probability of waking in sleeping mice. Importantly, disruption of either cluster can give rise to debilitating neurological disorder. Mutation of the hcrt locus, or loss of hcrt expressing neurons, results in the sleep disorder narcolepsy. Disruption of the LC has been implicated in Parkinson's and Alzheimer's diseases, Down and Rett syndromes, drug addiction, attention- deficit hyperactivity disorder and anxiety disorders. In vitro studies have provided strong evidence for interactions between Hcrt neurons and the LC. Hcrt neurons densely innervate the LC, LC neurons express the Hcrt receptor (HcrtR) and Hcrt can activate the LC in vitro. However, our understanding of the developmental, neuronal and behavioral interactions between these two systems in vivo is limited due to the large number of these neurons in mammals, their location deep within the mammalian brain, the complexity of their projections, the difficulty of imaging neural circuits in live mammals and the difficulty of analyzing mammalian behavior in a high-throughput fashion. Danio rerio (zebrafish) larvae are optically transparent and have only ~10 Hcrt and ~6 LC neurons per hemisphere. The mammalian pattern of hcrt expression and Hcrt neuron anatomy, activity and function are conserved in zebrafish. Furthermore, drugs that impinge upon the NA system similarly affect sleep/wake behaviors in zebrafish and mammals, suggesting that the function of the LC is conserved. This proposal exploits the advantageous features of zebrafish larvae to investigate Hcrt-LC interactions in development, neuronal activity and behavior. The development of Hcrt neuron projections to the LC and the arborization of these projections will be characterized at single neuron resolution in live animals using time-lapse microscopy. To test the hypothesis that LC neurons provide cues necessary for the normal extension and arborization of Hcrt projections, the LC will be ablated and silenced. To test the hypothesis that the LC provides cues that are sufficient to instruct Hcrt neuron morphogenesis, ectopic LC-like neurons will be generated. Hcrt overexpression, as well as optogenetic stimulation of Hcrt neurons, will be used to assess whether Hcrt neurons can drive LC activation in vivo. Hcrt overexpression in zebrafish larvae has been shown to increase locomotor activity, decrease the amount of time spent in a sleep-like state and induce hyperarousal. Cell ablation and neural silencing techniques will be used to assess the extent to which the LC mediates these phenotypes. These in vivo studies should yield insights into the developmental and functional interactions between two neuronal circuits of fundamental importance in sleep regulation and human health. PUBLIC HEALTH RELEVANCE: The hypocretin neurons of the hypothalamus and the locus ceruleus neurons of the brainstem form two major centers of sleep regulation. Disruption of either center is implicated in debilitating neurological disorders: hypocretin neurons are disrupted in narcoleptic patients, and the locus ceruleus is important in the development of Parkinson's and Alzheimer's diseases, Down and Rett syndromes, drug addiction, attention- deficit hyperactivity disorder and anxiety disorders. This proposal aims to investigate the physical connections between these two centers, how these connections are formed during development and how these systems interact with each other to regulate sleep/wake behaviors. !